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B    M   577   DOb 


1922 


A  Biochemical  Study  of  Streptococci 


With  Special  Emphasis  on  the  Determination  of 
Their  Chemical  Composition 


DISSERTATION 

Submitted  in  partial  fulfillment  of  the  requirements  for  the  degree  of 

Doctor  of  Philosophy  in  the  Faculty  of  Pure  Science  of 

Columbia  University 


By 
FRANCES  KRASNOW,  B.S.,  A.M. 


NEW  YORK  CITY 
1922 


A  Biochemical  Study  of  Streptococci 


With  Special  Emphasis  on  the  Determination  of 
Their  Chemical  Composition 


DISSERTATION 

Submitted  in  partial  fulfillment  of  the  requirements  for  the  degree  of 

Doctor  of  Philosophy  in  the  Faculty  of  Pure  Science  of 

Columbia  University 


By 
FRANCES  KRASNOW,  B.S.,  A.M 


NEW  YORK  CITY 
1922 


CONTENTS 

I.     Introduction 

1 .  General  Statement 

2.  Historical 

3.  Object  of  Research 

II.     Character  of  Organisms 

1 .  Source 

2.  Preparation  of  media  for  use  in  the  determination  of 
the  biochemical  reactions. 

(a)  Sugar-free  broth 

(b)  Carbohydrate  broth 

(c)  Meat  infusion  agar 

(d)  Meat  infusion  gelatin 

(e)  Milk 

(f)  Inulin — serum — water 

(g)  Bile 

3.  Preparation  of  the  reagents  for  the  determination  of 
the  hydrogen-ion  concentration. 

(a)  Standard  solutions 

(b)  Indicators 

4.  Isolation 

5.  Stock  Cultures 

6.  Seed  Cultures 

7.  Biology  and  Biochemistry  of  the  organisms 

(a)  Blood  reactions 

(b)  Immunity  reactions 

(c)  Other  biochemical  reactions 

8.  Description  of  the  test  organisms 

III.     Preparation  of  the  Mass  Cultures 
I .    Medium 

(a)  Choice  of  the  Infusion  basis 

(1)  Hemolyzed  blood- veal-infusion  agar 

(2)  Huntoon  agar 

(3)  Hasting  gelatin-agar 

(4)  Beef  liver  agar 

(5)  Beef  brain  agar 

(b)  Concentration  of  agar — agar 

(c)  Constancy  in  composition 


488835 


2.   Cultures 

(a)  Preparation  of  the  cultures  for  mass  growing 

(b)  Planting  of  cultures 

(c)  Removal  of  growth  from  agar 

(1)  Washing  method 

(2)  Spatula  method 

(d)  Drying  of  growth 

IV.     Analysis  of  the  Bacterial  Material 

1 .  Description  of  methods  employed 

(a)  Nitrogen 

(b)  Total  sulphur  and  phosphorous 

(c)  Ash 

(d)  Moisture 

(e)  Lipins 

(f)  Blanks 

2.  Records  of  analysis 

3.  Summary  of  the  data  for  the  average  percentage  composition 
of  the  streptococci 

4.  Conclusions  from  the  analytical  data 

V.     Analysis  of  the  Lipin  Mixture 

1 .  Outline  of  the  procedure 

2.  Records  of  analysis 

3.  Summary  of  the  data  for  the  average  percentages  of  phosphatide  and 
cholesterol. 

4.  Conclusions  from  the  data  on  the  cholesterol  and  phosphatide  content 

VI.     Effect  of  the  Lipin  Material  on  Hemolysis 

1 .  Record  of  experiments 

2.  Conclusions  from  the  data  on  hemolysis 

VII.     Summary  of  Conclusions 
VIII.     Bibliography. 


I.     INTRODUCTION 

1 .  GENERAL  STATEMENT:  The  chief  purposes  pervading  researches  on 
the  streptococci  are  two:  (i)  to  get  a  sound  basis  for  the  classification  of 
the  different  types  and  (2)  to  understand  the  role  they  play  in  the  general 
processes  of  nature.    All  the  results,  thus  far,  are  based  on  the  changes 
which  these  organisms  produce  in  their  environment.    Though  this  in- 
formation is  very  valuable  we  must  not  forget  that  in  order  to  compre- 
hend function  we  must  understand  structure  and  composition .    Discovery 
of  the  varieties  of  relation  of  the  actual  constituents  of  the  bacterial  bodies 
must  lead  to  a  better  realization  of  the  very  things  for  which  we  are  seek- 
ing.   With  this  purpose  in  view,  we  have  undertaken  the  work  to  be  de- 
tailed here. 

2.  HISTORICAL:  This  is  not  the  first  study  on  bacterial  composition. 
Thus  Brieger  as  early  as  1885  (i)  analyzed  pneumococcus  cultures;  Weyl 
(2)  and  Hammerschlag  in  1891  (3),  Hoffman  in  1894  (4)»  De  Schweinitz 
and  Dorset  in  1895  (5),  1896  (6),  1897  (7),  1898  (8),  Ruppel  in  1898  (9) 
and  Levene  in  1898  (10)  and  1901   (n)  investigated  the  chemistry  of 
Baccilus  Tuberculosis;  Drymount  in  1886  (12)  and  later  Vaughan  (13) 
and  Wheeler  (14)  worked  with  Bacillus  Anthrax.    More  recently  Vaughan 
(15),  Leach  (16)  and  Dawson  (17)  analyzed  Bacillus  Coli  cultures  and 
Nicholle  and  Alilaire  (18)  and  Bradley  (19)  used  Bacillus  Diphtheria  as 
their  material  for  analysis. 

3.  OBJECT  OF  RESEARCH:  Each  of  these  reports  supply  interesting 
additions  to  our  knowledge  of  the  chemical  make-up  of  bacteria.    It 
might,  therefore,  seem  that  our  problem  is  a  very  simple  one,  differing 
from  previous  investigation  only  in  respect  to  the  organism,  considered. 
Such  a  view  cannot  be  more  than  superficial.    Our  aim,  here,  is  not  merely 
to  gather  data  on  the  chemical  composition  of  the  streptococcus — it  is  to 
gather  such  data  under  standardized  conditions.    Practically  nowhere  in 
the  literature  is  there  mention  of  such  an  attempt,  though  it  is  precisely 
the  want  of  standard  methods  that  has  made  so  many  results  in  bacterio- 
logical work  of  little  or  no  value.     We  have,  therefore,  made  special 
effort  to  work  out  details.    This  will  become  apparent  with  a  careful 
consideration  of  the  methods  of  procedure  described  below: 

II.   CHARACTER  OF  ORGANISMS 

1.  SOURCE:   All  the  strains  of  streptococci  were  obtained  from   root 
canal  infections  in  the  teeth  of  man.* 

2.  PREPARATION  OF  MEDIA  FOR  USE  IN  DETERMINING  THE  BIOCHEMICAL 
REACTIONS: 

(a)  Sugar-free  broth:  75  grams  of  Bacto-veal  in  1000  cc.  of  tap  water 
were  heated  in  the  Arnold  sterilizer  for  two  hours  and  filtered  through 
filter  paper.  This  infusion  was  inoculated  with  B.  coli  communior  (20), 
incubated  at  37°  C.  for  18  hours  and  sterilized  at  "Arnold"  temperature 
for  45  minutes.  The  hot  bouillon  easily  dissolved  the  10  grams  of  peptone 
and  5  grams  NaCl  that  were  added  at  this  point.  An  aliquot  part  (locc.) 
was  cooled  and  adjusted  with  0.05  M  NaOH  to  the  hydrogen-ion  concen- 
tration, PH  =  7.9  (21).  The  calculated  amount  of  M  NaOH  was  added 

*A11  the  cultures  were  obtained  from  Dr.  M.  L.  Rhein  and  Dr.  J.  M.  Levy,  New 
York  City. 


to  the  remainder  and  set  in  the  Arnold  sterilizer  for  15  minutes.  Such 
treatment  usually  results  in  an  increased  acidity,  the  PH  changing  to  a 
value  between  7.7  and  7.5.  This  preparation  had  a  final  PH  =  7.7.  The 
medium  was  filtered  and  sterilized  in  the  "Arnold"  for  30  minutes  on  three 
successive  days. 

(b)  Carbohydrate  broth:  The  chief  point  to  guard  against  in  this  connec- 
tion is  that  a  sugar  in  slightly  alkaline  solution  decomposes  readily.    This 
occurs  to  some  extent  even  at  room  temperature  (23°  to  25°  C.)  but  very 
marked  decomposition  sets  in  at  the  temperature  of  the  Arnold  sterilizer. 
It  is  best,  therefore,  to  sterilize  a  concentrated  (20  per  cent)  aqueous  so- 
lution of  the  carbohydrate  by  boiling  (10  to  15  minutes  being  sufficient) 
(20).    This  concentrated  solution  is  then  mixed  with  sterile    sugar-free 
broth  in  amounts  to  make  the  desired  dilution.    The  sterility  of  the  mix- 
tures was  tested  by  incubating  them  for  18  hours  at  37°  C. 

(c)  Meat  infusion  agar:  The  basis  for  this  medium  was  made  by  in- 
fusing 75  grams  Bacto-veal  in  500  cc.  tap  water  in  the  "Arnold"  for  two 
hours.      The  steps  in  the  procedure  for  obtaining  the  finished  bouillon 
product  parallelled  those  for  sugar-free  broth  with  the  exception  that 
there  was  no  inoculation  with  B.  coli.    To  this  substrate  was  added  an 
equal  quantity  of  a  4  per  cent  solution  of  agar-agar,  giving  a  final  con- 
centration equal  to  2  per  cent. 

(d)  Meat  infusion  gelatin:    Hiss  and  Zinsser  recipe  (22). 

(e)  Milk:  Hiss  and  Zinsser  recipe  (23). 

(f)  Hiss  inulin-serum-ivater :  Hiss  and  Zinsser  recipe  (24) . 

(g)  Bile:  Ox-bile  obtained  from  the  slaughter  house  was  sterilized  for 
20  minutes  on  three  successive  days. 

3.  PREPARATION  OF  THE  REAGENTS  USED  IN  THE  DETERMINATION  OF 
HYDROGEN-ION  CONCENTRATIONS:  In  determining  hydrogen-ion  concen- 
trations the  colorimetric  method  was  chosen.  The  principles  involved 
need  not  be  reiterated  here;  they  have  been  thoroughly  described  by  Clark 
and  Lubs  (25).  The  chief  sources  of  error,  color  of  medium  and  tubidity 
of  culture  were  obviated  by  diluting  the  medium  (one  part  medium  to  two 
parts  distilled  water)  and  then  compensating  for  the  color  by  Walpole's 
comparator  method  of  superimposing  the  color  of  the  medium  upon  that 
of  the  indicator  (26,  27). 

(a)  Standard  solutions  used:    i  /I5  M  disodium  hydrogen  phosphate 

I  /i  5  M  potassium  dihydrogen  phosphate 
I  /5  M  sodium  acetate 
i  /5  M  acetic  acid 

The  salts  were  recrystallized  repeatedly  until  they  satisfied  Sorensen's 
purity  standards  (28).  The  acetic  acid  was  obtained  by  redistilling  C.P. 
glacial  acetic  acid.  The  phosphate  mixtures  were  prepared  by  Sorensen's 
technique  and  the  acetic  acid-sodium  acetate  mixtures  according  to  Wal- 
pole's directions  (26).  The  accuracy  of  these  standard  mixtures  was 
verified  by  the  hydrogen  electrode.*  The  actual  values  obtained  prac- 
tically duplicate  those  given  by  these  authors.  A  study  of  the  following 
table  will  make  this  evident. 

*We  are  indebted  to  Dr.  Hastings,  of  the  Rockefeller  Institute,  New  York  City,  for 
aid  in  this  connection. 


Author 

Mixtures  in  c.c. 

PH 

Walpole 

CHsCOONa 

CH3COOH 

Given 

Obtained 

5-0 

5-0 

4.62 

4-63 

5-0 

7-o 

4.27 

4-34 

Sorensen 

Na2HPO4 

KH2PO4 

9.0 

1.0 

7.64 

7-57 

5-0 

5-0 

6.81 

6.78 

These   figures   were   chosen   as   examples   because    the    test   materials 
matched  these  most  often  in  the  colorimetric  determinations. 

(b)  Indicators  used:  The  indicators  used  were  those  recommended  by 
Clark  and  Lubs  and  manufactured  by  Hynson,  Westcott  and  Dunning, 
Baltimore.  The  following  table  shows  the  character  of  the  indicator 
solutions  and  notes  the  amounts  of  the  several  varieties  used: 


Name 

Concentra- 
tion 
of  Solution 

Solvent 

Amount 
used  per 
10  c.c. 

Range 
of 
PH 

Tetabromphenolsulphonephtha- 
lein  (Brom  phenol  blue) 

0.04% 

95% 
alcohol 

0.3  c.c. 

2.8-4.6 

Orthocarbobenzeneazodimethyl- 
anilin    (Methyl  red) 

0.02% 

water 

0.5  c.c. 

4.4-6.0 

Dibromocresolsulphonephthalein 
(Brom  cresol  purple) 

0.04% 

95% 
alcohol 

0.3  c.c. 

5.2-6.8 

Phenolsulphonephthalein 
(Phenol  red) 

0.02% 

95% 
alcohol 

0.3  c.c. 

6.8-8.4 

4.  ISOLATION  OF  THE  STRAINS:  The  fresh  material  taken  from  the  root 
canal  was  planted  on  hemolyzed  sheep  blood-glucose-veal  infusion  agar 
slants  (0.2  c.c.  sterile  hemolyzed  defibrinated  blood  plus  0.5  c.c.  12% 
sterile  glucose  solution  for  each  6  c.c.  medium)  to  which  o.i  c.c.  sterile 
i%  glucose-beef-infusion  broth  had  been  added  after  solidification. 
After  1 8  to  20  hours  incubation  at  37°  C.,  a  loopful  was  streaked  on  several 
whole  sheep-blood  glucose  infusion  agar  plates.  Single  colonies  fished 
from  these  plates  were  inoculated  into  glucose  infusion  broth.  If  micro- 
scopic examination  proved  this  a  pure  streptococcus  culture,  transplants 
were  made  to  whole  sheep  blood  infusion  agar  (0.5  c.c.  blood  plus  9.5  c.c. 
agar)  and  ox  bile.  The  reactions  obtained  for  70  cultures  used  as  stock 
strains  are  given  in  the  following  table: 


Strain 
852 

853 
863 
874 

877 
882 
894 
904 
909 
911 
922 
948 
953 
956 
961 


Reaction  to 


Whole  Blood 
I 
I 

G 
I 
I 
I 
I 

H 
I 
I 
I 
I 

G 
I 
I 


Bile 

Strain 

O 

981 

0 

1007 

O 

1009 

0 

IOI2 

0 

1015 

0 

1028 

O 

1041 

0 

1044 

O 

1074 

0 

1081 

O 

1094 

O 

1148 

0 

1151 

O 

H54 

O 

1155 

Reaction  to 
Whole  Blood 
I 

I 
I 
G 

I 

I 

I     . 

G 

G 

I 

I 

I 

G 

G 

I 


Bile 
O 


Bile 

Strain 

O 

777 

0 

802 

O 

804 

0 

830 

O 

838 

0 

898 

O 

907 

0 

910 

O 

942 

O 

947 

O 

948 

0 

966 

O 

978 

O 

1004 

O 

1010 

O 

IOII 

O 

1060 

O 

1097 

0 

3 

O 

4 

Reaction  to 

Whole  Blood 

Bile 

G 

O 

I 

0 

I 

0 

I 

0 

I 

O 

I 

O 

I 

O 

I 

0 

G 

0 

I 

O 

I 

0 

G 

0 

I 

0 

I 

O 

H 

0 

H 

0 

I 

O 

G 

O 

I 

0 

I 

0 

Reaction  to 

Strain  Whole  Blood 

1159  G 

1161  I 

I2IO  H 

1220  G 

1221 

1232 

1236 

1239 

1279  G 

1286  I 

1293  G 

682  I 

684  I 

688  I 

703  G 

720 

725  I 

746 

748 

770  I 

O  =  No  solution  of  organisms  by  bile.  ^ 

I  =  Indifferent  to  blood  cells. 
H  =Hemolysis  of  blood  cells. 
G  =  Green-producing  action  on  blood  cells. 

5.  STOCK  CULTURES:  After  isolation,  each  organism  was  planted  in 
2  c.c.  defibrinated  sheep  blood,  incubated  for  four  hours  at  37°  C.  and 
then  stored  in  the  ice-chest  at  10°  C.  Some  of  the  strains  were  used  after 
several  days  isolation,  others  after  several  months  of  cultivation. 

6.  SEED  CULTURE:    Two  days  before  a  specific  culture  had  to  be  used, 
a  loopful  (platinum  loop,  4mm.  in  diameter)  from  the  stock  blood  culture 
was  planted  in  i%  glucose  broth  (9.5  c.c.  sugar-free  broth  plus  0.5  c.c. 
sterile  20  per  cent  glucose  solution  and  incubated  for  18  hours  at  37°  C. 
Then  0.05  c.c.  of  this  subculture  was  inoculated  into  plain  infusion  broth 
(not  made  sugar-free)  and  incubated  for  the  same  period.    This  is  desig- 
nated as  a  seed  culture. 

In  order  to  insure  uniformity  in  the  number  of  organisms  transplanted 
from  such  seed  cultures  to  the  test  media  it  was  necessary  to  estimate 
the  amount  of  growth.  The  cultures  were  well  shaken  and  diluted  to 
equal  opacity  by  matching  in  a  comparator  block  (the  kind  used  in  the 
colorimetric  determination  of  hydrogen-ion  concentration).  Then  the 
accuracy  of  this  procedure  was  verified  by  the  results  obtained  from 
plating  definite  amounts  of  the  matched  cultures  and  counting  the  colonies 
after  18  hours  incubation  at  37°  C.  For  all  comparative  tests  these 
matched  cultures  served  as  the  seeds. 

7.  BIOCHEMISTRY    AND    BIOLOGY: 

(a)  Reaction  on  blood  medium:  The  significance  of  this  reaction  may 
be  clearly  brought  out  by  a  brief  review  of  the  literature  on  the  classi- 
fication of  the  streptococci.  At  the  very  outset  investigators  attempted 
to  differentiate  these  organisms  according  to  morphological  characters. 
Thus  arose  the  groups  (i)  Streptococcus  longus  and  (2)  Streptococcus 
brevis  (29,  30) .  Such  a  distinction  between  types  could  be  nothing  more 
than  tentative  and  therefore  was  short-lived. 

Collateral  with  this  grouping,  but  of  more  lasting  influence,  there 
existed  another — that  based  on  source  of  origin.  We  refer  here  to  such 
groups  as  Streptococcus  erysipelas  (31,  32),  Streptococcus  equinus, 
Streptococcus  pyrogenes,  Streptococcus  salivarans,  etc.  (33,  34,  35). 
This  scheme  too,  could  have  but  little  value  for  there  would  be  as  many 


8 


groups  of  streptococci  as  there  are  places  from  which  these  organisms  may 
be  isolated. 

Then  Schottmiiller  (36)  suggested  a  new  classification  into  strepto- 
coccus erysipelas,  Streptococcus  viridans  and  Streptococcus  mucosus,  va- 
rieties recognized  by  certain  distinctive  appearances  on  blood  media. 
While  this  method  was  gaining  ground  in  Germany,  English  bacteriolo- 
gists were  studying  the  fermentation  reactions  of  streptococci  in  order  to 
ascertain  whether  essential  differences  could  be  discovered .  In  this  con- 
nection the  researches  of  Gordon  (33)  and  Andrews  and  Horder  (34) 
are  significant. 

Still  another  basis  for  classifying  the  streptococci  has  been  considered — 
that  involving  the  immunity  reactions  (35,  37-47).  Most  investigators 
think  these  reactions  parallel  the  blood  reactions  (43, 44,  45) ,  while  Kligler 
(35)  seems  to  be  at  variance  with  such  a  conclusion.  From  his  experi- 
ments he  finds  a  closer  relationship  between  strains  fermenting  the  same 
carbohydrates  than  between  strains  having  the  same  hemolytic  power. 

More  recently  investigations  have  introduced  nothing  new  so  far  as 
fundamental  principles  of  classification  are  concerned.  All  of  them 
treat  with  one,  or  with  a  combination  of  the  bases  outlined  above.  Various 
schemes  have  been  proposed.  Most  attention  has  been  given  to  those 
which  make  use  of  the  blood  reaction.  We  may  mention  here  the  work  of 
Holman  (48),  Blake  (49)  and  Smith  and  Brown  (45).  These  authors 
recognize  two  main  types — hemolytic  and  non-hemolytic,  divided  into 
subgroups  which  depend  on  the  fermentative  power  of  the  organisms. 
And  finally,  the  scheme  which  has  superceded  all  of  these  and  is  now 
considered  as  the  scheme  by  some  of  the  most  prominent  bacteriologists 
is  based  primarily  on  the  blood  reaction  and  includes  three  main  groups. 
Reference  is  made  to  the  work  of  Lyall  (50),  Brown  (51)  and  Park  and 
Williams  (52). 

A  thorough  consideration  of  the  various  suggestions  has  led  also  us  to 
conclude  that  the  best  basis  for  a  preliminary  grouping  of  the  strepto- 
cocci is  that  last  named.  The  three  types  recognized  are  (i)  that  which 
hemolyzes  the  blood  corpuscles;  (2)  that  which  produces  a  green  colora- 
tion; (3)  that  which  is  indifferent  to  the  presence  of  blood. 

Detailed  technique  for  differentiating  the  "blood  types": — 0.05  cc. 
culture  from  seed  tube  was  dropped  on  the  blood  agar  (0.5  cc.  blood 
plus  9.5  cc.  agar)  in  a  petri  dish  and  spread  over  the  entire  surface  with  a 
special  smooth  glass  rod.  The  depth  of  the  agar  was  2  mm.  Precaution 
was  taken  to  have  the  blood  evenly  distributed.  The  results  recorded 
were  obtained  from  the  examination  of  colonies  widely  separated  after 
24  hours  incubation  at  37°  C. 

In  view  of  the  fact  that  different  laboratories  use  blood  from  different 
animal  species  we  felt  it  necessary  to  find  out  whether  the  various  strains 
reacted  alike  on  the  bloods  most  commonly  used,  human,  rabbit  and 
sheep.  As  might  have  been  expected,  the  response  of  the  organisms  to 
the  different  kinds  of  erythrocytes  was  not  the  same.  The  actual  ex- 
perimental findings  for  70  cultures  are  as  follows: 


Strain 

Reaction  to 
Human  Blood               Rabbit  Blood                 Sheep  Blood 

H 

I 

G 

H 

I 

G 

H 

I 

G 

852 
853 
863 

877 
874 

+ 
+ 

+ 
+ 

+ 

+ 

+ 

+ 
+ 

+ 

+ 
+ 

+ 
+ 

+ 

Strain 

Reaction  to 
Human  Blood       Rabbit  Blood       Sheep  Blood 

H 

I 

G 

H 

I 

G 

H 

I 

G 

882 

+ 

T 

, 

894 

+ 

+ 

4. 

904 

+ 

4,- 

+ 

909 

+ 

4r 

^ 

9" 

4- 

4- 

4. 

922 

+ 

4. 

_i_ 

948 

-f 

+ 

4. 

953 
956 

+ 

+ 

_,_ 

+ 

+ 

961 

+ 

+ 

i 

981 

4- 

4r 

4, 

1007 

4- 

+ 

^ 

1009 

4- 

4- 

4. 

IOI2 

+ 

4^ 

i 

1015 

+ 

^ 

+ 

1028 

4- 

4^ 

IO4I 

+ 

+ 

^_ 

1044 

+ 

+ 

i 

1074 
1  08  1 

+ 

+ 

+ 

+ 

+ 

+ 

1094 

4- 

4^ 

+ 

1148 

+ 

+ 

"54 

+ 

_|_ 

+ 

+ 

"55 

+ 

4. 

i 

"59 

IIDI 

| 

t 

+ 

I2IO 

4r 

4^ 

i 

I22O 

^_ 

_j_ 

, 

1221 

4^ 

_j_ 

i 

1232 
1236 

| 

+ 

+ 

1239 

4- 

4,- 

i 

1279 

4^ 

_L 

i 

1286 

^ 

i 

i 

1293 

4r 

4. 

i 

682 

4. 

_j_ 

i 

684 

+ 

4. 

T 

688 

4r 

i 

T 

703 

4^ 

_)_ 

i 

720 

-{- 

4^ 

i 

725 

4- 

_j_ 

i 

746 

4^ 

_j_ 

i 

748 

+ 

4. 

i 

770 

+ 

4^ 

i 

777 

4- 

_|_ 

i 

802 

4^ 

i 

' 

804 

4- 

i 

830 

4- 

i 

i 

838 

4^ 

_i_ 

T 

898 

4- 

4^ 

i 

907 

4- 

_(_ 

i 

910 

4- 

_j_ 

i 

942 

4- 

_|_ 

i 

947 

4- 

_i_ 

i 

948 

4^ 

_j_ 

T 

966 

4. 

_j_ 

, 

978 

+ 

4^ 

_l_ 

1004 

-\- 

^ 

i 

IOIO 

-\- 

4_ 

i 

IOII 

1060 

+ 

| 

+ 

+ 

+ 

- 

1097 

4. 

J. 

i 

3 

+ 

+ 

_l_ 

4 

Note:  H=hemolysis.      I  =  indifferent.      G  =  green  producing. 


10 


These  results  were  checked  by  triplicate  determinations. 

The  above  detailed  tabulation  may  be  summarized  as  follows:- 


No.  of  Strains 

No.  of  Strains 

No.  of 

Source  of  Blood 

Producing 

Producing 

Indifferent 

Hemolysis 

Green  Coloration 

Strains 

Man 

16 

7 

47 

Rabbit 

17 

7 

46 

Sheep 

4 

16 

50 

No.  of  "fast"  strains 

4 

7 

40 

Note:  A  "fast"  strain  is  one  which  reacts  alike  to  the  blood  of  all  species. 

This  table  indicates  definitely  the  probable  source  for  many  discrep- 
ancies. It  indicates,  too,  that  a  very  decided  advance  in  the  systematic 
study  of  the  streptococci  may  be  made  by  instituting  definite  standards 
of  procedure. 

The  group  of  organisms  used  for  analytical  material  included  two  "fast1' 
strains  of  each  type. 

(b)  Immunity  reactions:  These  reactions  were  studied  in  order  to 
demonstrate  that  the  chosen  organisms  were  biologically  distinct.  To 
prove  this  point,  the  agglutinative  capacity  of  each  antiserum  against 
each  of  the  antigens  was  determined.  The  method  of  producing  the 
immune  sera  and  the  technique  for  conducting  the  titrations  was  that 
used  by  Dochez,  Avery  and  Lancefield  (53). 

The  results  obtained  (as  may  be  seen  from  the  table)  show  no  duplica- 
tion in  the  strains  used. 


Immune  Serum 


904 

I2IO 

863 

1074 

688 

72O 

904 

1-500 

I-IOO 

0 

0 

o 

0 

I2IO 

1-150 

1-450 

0 

0 

0 

0 

863 

0 

0 

1-350 

1-250 

0 

0 

1074 

0 

0 

1-200 

1-650 

0 

o 

688 

1-50 

0 

1-50 

1-30 

I-20O 

I-IOO 

720 

0 

I-IO 

I  -2O 

I-2O 

1-50 

1-550 

Note:  The  numbers  under  "immune  serum"  designate  the  highest  dilutions  of  the 
antiserum  which  showed  definite  specific  clumping  of  the  organisms. 

(c)  Other  biochemical  reactions:  Although  our  choice  of  the  experi- 
mental strains  depended  almost  entirely  on  the  blood  reactions  yet  a 
complete  characterization  of  the  organisms  must  include  the  description 
of  their  ability  to  (i)  ferment  various  carbohydrates  and  related  sub- 
stances (33,  34);  (2)  coagulate  milk  (33,  34);  (3)  coagulate  inulin-serum- 
water  (53,  54);  (4)  liquefy  gelatin  (34);  (5)  dissolve  in  bile  (55). 

The  test  substance  (10  c.c.)  in  each  series  (except  5)  was  inoculated 
with  0.05  c.c.  culture  from  the  seed  tubes.  In  series  (5)  0.3  c.c.  bile 
was  mixed  with  3  c.c.  culture.  All  except  series  (3)  were  incubated  at 
37°  C.  for  24  hours.  Series  (3)  was  incubated  for  6p  hours.  The  follow- 
ing tabulation  lists  the  reactions  given  by  the  experimental  cultures: 


n 


c 

Hydrogen-ion  Concentration  , 

Reaction  in 

o 

PH,  in  Broth 

B 

, 

Strain 

Blood  Reaction 

£ 

imunitj 

s 
•«* 

13 

<a 
tf 

H 

8 

8 

M 

<a 

to 

_ft 

^ 

to 

_ft 

<0 

to 

_ft 

"3 

crose 

« 
«•> 

o 
S 

£ 

s 

:§ 

-2 

C 

'•2 

« 

e-S 
J^ 

b 

* 

O 

* 

% 

t^ 

% 

•5 

3 

§ 

5 

1" 

Control 

7-7 

7-7 

7-7 

7-7 

7-7 

7-7 

7-7 

7.7 

7-7 

7-7 

o 

o 

o 

o 

904 

Hemolysis 

+ 

7-7 

4.8 

6.2 

4.8 

7-7 

4-3 

4.6 

5-9 

5-0 

7-7 

0 

-f 

o 

0 

1210 

Hemolysis 

-j- 

7-7 

4.6 

5-9 

4.6 

4.6 

4-3 

6.8 

5-9 

4.6 

7-7 

0 

-f- 

o 

o 

863 

Green-producing 

•f- 

7-7 

4.6 

7-7 

4.6 

4.6 

4-3 

7-7 

6-5 

7-7 

7-7 

o 

-f- 

0 

o 

1074 

Green-producing 

-j- 

7-7 

4-3 

6-5 

4.6 

5-3 

4-3 

5-3 

6.2 

4.6 

7-7 

o 

-(- 

0 

o 

688 

Indifferent 

-f- 

7-7 

4.6 

5-6 

4.6 

4.6 

5-0 

7-7 

6.2 

4-3 

7-7 

0 

-f- 

o 

o 

720 

Indifferent 

+ 

7-7 

4-3 

6-5 

4.6 

4.6 

4-3 

4.8 

6-5 

4-3 

7-7 

o 

+ 

o 

o 

Note:     In  the  column  under  "immunity"  the  "  +"  sign  designates  that  the  culture  is 
biologically  distinct. 

8.  DESCRIPTION  OF  THE  TEST  ORGANISMS:  Six  test  organisms  were 
used:  two  hemolytic  strains,  two,  green-producing  and  two,  indifferent. 
The  members  of  each  pair  were  not  duplicated  individuals  of  the  same 
type  (see  carbohydrate  reactions).  Moreover  there  was  no  duplication 
of  strain  in  the  entire  series  (see  agglutination  reactions) . 

III.  PREPARATION  OF  MASS  CULTURES 
i.  MEDIUM: 

(a)  Choice  of  Infusion  basis:  A  comparative  study  of  the  different  solid 
media  available  for  streptococcus  cultivation  shows  that  the  organisms 
accumulate  more  rapidly  on  some  than  on  others.  Five  media  were 
considered . 

(1)  Hemolyzed  blood  glucose  veal  infusion  agar:    The  veal  infusion 
was  prepared  as  described  above  diluted  with  an  equal  volume 
of  3  per  cent  agar-agar  solution  and  sterilized.    When  needed, 
the  agar  was  melted  and  cooled  slowly  to  43°-45°  C.     Sterile 
hemolyzed  defibrinated  blood  (i  volume  blood  to  2  volumes  of 
distilled  water)   and   sterile   12%   glucose  solution  were   then 
added.    The  proportion  used  was  o.i  c.c.  of  the  diluted  blood 
and  0.25  c.c.  of  the  sugar  solution  to  every  3.0  c.c.  agar. 

(2)  Huntoon  agar  (56) . 

(3)  Hasting  gelatin-agar  (57):    The  original  directions  are  somewhat 
vague.    This  description  is  our  interpretation:    One  pound  beef 
meat  in  loooc.c.  water  was  boiled  for  one  hour,  strained  through 
a  wire  sieve,  made  up  to  the  original  volume,  10  grams  peptone 
and  5  grams  NaCl  added  and  set  in  the  Arnold  sterilizer  for  20 
minutes.    The  infusion  thus  obtained  was  used  as  the  solvent  for 
the  15  grams  agar-agar  and  20  grams  gelatin  added  at  this  point. 
Finally,  enough  NaOH  was  added  to  make  the  mixture  neutral  to 
phenolphthalein,  the  medium  being  kept  at  the  boiling  point 
during  the  titration. 

(4)  Beef  liver  agar:    500  grams  fresh  hashed  beef  liver  were  infused 
with  500  c.c.  tap  water  for  three  hours  at  55°  C.,  strained  through 
a  wire  sieve,  10  grams  peptone  and  5  grams  NaCl  added  and  set 
in  the  "Arnold"  until  solution  was  complete.    The  hydrogen-ion 
concentration  was  adjusted  to  a  PH  value  =  to  7. 9.    After  being 
mixed  with  500  c.c.  3  per  cent  agar-agar  solution,  the  infusion  was 


sterilized  in  the  autoclave  for  15  minutes  at  15  pounds  pressure. 

(5)  Beef  brain  agar:  This  preparation  was  made  exactly  as  the  beef 
liver  agar  except  that  brain  tissue  was  substituted  for  liver  tissue. 

Thus  prepared,  the  five  media  were  compared  by  this  procedure:  Each 
medium  was  tested  with  each  experimental  culture.  We  had,  therefore, 
six  series  of  tests — one  for  each  strain.  0.05  c.c.  of  culture  from  the  several 
seed  tubes  were  evenly  distributed  over  the  surfaces  of  agar  slants  of  the 
respective  media.  All  the  slants  were  formed  by  equal  amounts  of 
medium.  Readings  were  made  after  1 8  hours  incubation  at  37°  C.  The 
data  tabulated  below  indicate  the  results  obtained  for  one  series.  They 
are  representative  since  each  series  showed  the  same  gradations  in  the 
amounts  of  growth. 


Medium 

Estimated  Amount  of  Growth 

Hemolyzed  blood  agar 
Huntoon  agar 
Hasting  gelatin-agar 
Brain  agar 
Liver  agar 

Abundant 
Abundant 
Slight 
Slight 
Very  abundant 

+  +  + 
+  +  + 
+  + 
+  + 
+  +  +  + 

The  results  are  necessarily  only  comparatively  quantitive.  They 
indicate,  however,  that  the  liver  infusion  furnished  the  optimum  material 
for  maximum  growth.  This  may  be  explained  by  the  fact  that  the  liver 
normally  contains  a  large  store  of  glycogen,  amino  acids,  various  secre- 
tions, etc.,  all  probably  valuable  to  bacterial- nutrition. 

(b)  Concentration  of  agar-agar:  The  more  concentrated  the  agar- 
agar  in  a  medium,  the  greater  is  the  insurance  against  scratching  the 
surface  of  a  solidified  layer  in  a  petri  dish  when  removing  growth  with  a 
smooth  spatula.  But,  on  the  other  hand,  too  concentrated  a  preparation 
will  practically  inhibit  bacterial  growth.  It  therefore,  became  necessary 
to  find  that  concentration  which  permitted  the  maximum  development  of 
colonies  and  at  the  same  time  insured  against  the  possible  breaking  of 
the  surface.  Six  series  of  tests  were  performed,  one  for  each  experimental 
culture.  The  infusion  subtrate  was  the  same  for  all;  the  concentration  of 
the  agar-agar  was  varied  for  each  series.  The  results  show  definitely  that 
for  the  organisms  used  2  per  cent  is  the  optimum  concentration. 


Percent.  || 
agar-agar  || 

Growth  for  the  Various  Organisms 

Streaking 
Character 

Removal 
of 
Growth 

688 

720 

863 

1074 

904 

I2IO 

I.O 

+  +  +  + 

+  +  +  + 

+  +  +  + 

+  +  +  + 

+  +  +  + 

+  +  +  + 

Unfavor- 

Impos- 

able 

sible 

i-5 

+  +  +  + 

+  +  +  + 

+  +  +  + 

+  +  +  + 

+  +  +  + 

+  +  +  + 

Difficult 

Difficult 

2.O 

+  +  +  + 

+  +  +  + 

+  +  +  + 

+  +  +  + 

+  +  +  + 

Very 

Very 

favorable 

Favorable 

2-5 

+  +  + 

+  +  +  + 

+  +  + 

+  +  + 

+  +  +  + 

+  +  + 

Very 

Very 

favorable 

avorable 

3-0 

+  + 

+  + 

+  +  + 

+  + 

+  +  + 

+  + 

Very 

Very 

favorable 

avorable 

3-5 

+  + 

+  + 

+  + 

+  + 

+  +  + 

+  + 

Very 

Very 

favorable 

avorable 

4.0 

+ 

+ 

+  + 

+ 

+  + 

+ 

Very 

Very 

favorable 

avorable 

4-5 

+ 

+ 

+ 

± 

+  + 

+ 

Very 

Very 

favorable 

avorable 

5-° 

+ 

± 

+ 

+ 

+ 

+ 

Very 

Very 

favorable 

avorable 

(c)  Constancy  in  composition  of  medium:  To  insure  absolute  constancy 
in  the  composition  of  the  medium  a  stock  infusion  was  prepared  from  one 
large  shipment  of  fresh  hashed  liver  (200  pounds).  This  was  estimated 
as  the  quantity  large  enough  to  cover  the  needs  of  the  entire  investigation. 

2.  CULTURES: 

(a)  Preparation  of  cultures  for  mass  growing:  Jablons  liver  peptone 
broth  (58)  was  used  for  the  cultivation  of  the  test  organisms  preliminary 
to  mass  growing.  This  medium  attracted  our  attention  because  it 
contained  the  same  ingredients  as  the  solid  medium  selected,  thus 
favoring  exceptional  uniformity  in  the  metabolic  reactions  of  the  bacteria 
throughout  the  entire  experimental  period.  That  the  bouillon  supported 
hardy  development  may  be  proven  by  the  following  experiments  com- 
paring it  with  other  good  liquid  media:  (i)  Huntoon  broth  (56)  and  (2) 
Rosenow  broth  (59).  The  test  media  were  inoculated  with  one  loopful 
of  culture  from  the  seed  tubes,  kept  at  room  temperature  for  60  hours 
and  then  incubated  for  24  hours  at  37°  C.  Tests  were  made  on  50  strains 
of  streptococci.  A  comparison  of  the  amounts  of  growth  in  the  different 
preparations  will  give  convincing  proof  that  Jablons  medium  favors  rapid 
growth  of  the  organisms  under  consideration.  The  results  tabulated 
represent  the  reactions  given  by  all  the  organisms. 


Medium 

Amount  of  Growth 

Estimate 

Description 

Jablons  Broth 
Rosenow  Broth 
Huntoon  Broth 

+  +  +  + 
+  +  +  + 
+  +  + 

Very  abundant 
Very  abundant 
Quite  abundant 

At  regular  intervals  transplants  to  glucose  blood  agar  were  made, 
incubated  for  24  hours  at  37°  C.  and  examined  for  growth.  If  growth  was 
slight  or  doubtful,  subcultures  were  made  to  confirm  the  results.  By 
this  procedure  further  evidence  was  obtained  regarding  the  quality  of 
the  liver  broth.  A  mere  glance  at  the  following  summary  shows  that,  in 
addition  to  supporting  very  rapid  growth,  Jablons  medium  also  supports 
very  prolonged  viability. 


Medium 

No.  of  Cultures  Dead  in  Specified 
No.  of  Weeks  After  Inoculation 

Per  cent. 
Viable  After 

4 

6 

IO 

10  Weeks 

Jablons  Broth 

o 

o 

o 

IOO 

Rosenow  Broth 

o 

i 

2 

96 

Huntoon  Broth 

o 

i 

3 

94 

These  results  place  each  medium  in  the  order  of  preference  for  the 
cultivation  of  streptococci,  if  longevity  without  transplant  is  taken  as 
the  criterion.  With  four  weeks  as  the  limiting  period  no  differences  are 
noticed;  the  three  preparations  match  each  other  exactly.  But,  more 
prolonged  viability  necessarily  infers  less  interference  with  normal 
metabolism.  This  makes  Jablons  medium  the  most  efficient  since  it  is 
so  constructed  as  to  give  the  organisms  the  greatest  chance  for  continued 
vitality. 

After  growing  the  organisms  in  this  broth  for  one  month,  0.05  c.c. 
was  transplanted  to  liver  agar  plates  and  grown  at  37°  C.  for  18  hours. 
From  this  two  successive  subcultures  to  solid  liver  medium  were  made  on 
two  consecutive  days.  The  last  culture  served  as  the  seed  culture  for 
mass  planting. 

14 


The  mass  cultures  were  grown  for  16-18  hours  at  37°  C.,  thus  producing 
cultures  that  contained  a  minimum  of  degenerated  cells.  Of  course, 
this  procedure  did  not  supply  the  maximum  amount  of  growth  from  each 
planting,  but  it  did  supply  the  optimum  material  for  the  chemical  analysis 
of  the  normal  organisms.  This  point  seems  to  have  been  entirely  over- 
looked in  previous  investigations  on  bacterial  composition.  In  these, 
the  chief  interest  was  to  procure  the  maximum  amount  of  material  from 
each  planting.  No  attention  was,  therefore,  paid  to  the  metabolic  plane 
of  the  organism.  Thus  Brieger  (i)  used  growth  four  weeks  old,  Leach 
(60)  worked  with  cultures  one  to  two  weeks  old,  and  Vaughan  (15), 
stating  no  definite  period,  gathered  the  growth  when  it  had  reached  the 
maximum. 

(b)  Planting  of  Cultures:  The  agar  was  allowed  to  cool  slowly  to  43°- 
45°  C.,  thus  insuring  a  minimum  volume  of  water  of  condensation  in  the 
petri  dishes  after  solidification.    This  is  a  necessary  precaution  since  in 
too  moist  a  chamber  the  growth  becomes  saturated  with  moisture.    Such 
a  condition  is  very  unfavorable  for  the  purpose  at  hand,  because  (i)  it  is 
impossible  to  remove  any  significant  part  of  the  bacterial  masses  from 
the  agar  surface  and  (2)  the  small  fraction  that  is  obtained  is  contami- 
nated with  diffused  substance  from  the  medium. 

The  depth  of  the  agar  layer  in  the  plates  was  4mm. 
Transplants  were  made  from  the  special  seed  plates  described  above 
by  means  o   a  bent  smooth  glass  rod. 

(c)  Removal  of  the  growth  from  the  agar:  Two  possible  procedures  sug- 
gested themselves.    One  method  involves  the  detachment  of  the  growth 
from  the  subjacent  agar  with  bent  glass  rods,  simplifying  its  removal  by 
washing  with  physiological  salt  solution  and  pipetting  off  the  bacterial 
suspension  (15,  16).     It  is  evident  that  a  maximum  amount  of  material 
may  be  obtained  in  this  way  but  it  is  not  optimum  material  for  the  present 
study.     Much  of  the  soluble  substances  in  the  medium  are  washed  into 
the  salt  solution.      Then,   too,  some  substances  diffuse  from  the   cell 
bodies  into  the  outer  liquid.    Such  material  may  be  removed  by  several 
washings  and   centrifugations,   each    time  discarding   the  supernatant 
fluid.      By  doing  so,  however,  the  diffusible  bacterial  constituents  are 
continually  being  drawn  upon. 

The  other-method,  although  not  as  economical  regarding  the  collection 
of  material,  is  far  superior  in  providing  a  pure  mass  consisting  only  of 
"intact"  bacterial  structures.  This  process  is  simpler  and  involves 
fewer  manipulations  than  the  first.  Thus,  the  smoothened  end  of  a 
microscope  slide  is  held  lightly  but  firmly  on  the  agar  surface  and  rotated 
slowly  clockwise.  At  the  same  time  the  dish  is  turned  slowly  counter- 
clockwise. The  growth  is  thereby  gathered  on  the  slide  and  very  easily 
tapped  off  into  a  thoroughly  dried  shallow  glass  dish. 

In  order  to  determine  definitely  the  method  best  suited  for  our  purposes, 
tests  were  made  on: 

(1)  The  supernatant  NaCl  solution  after  centrifugati  on  of  thebacter- 
suspension,  obtained  by  removing  the  growth  from  the  agar  with  NaCl 
solution. 

(2)  The  supernatant  fluid  after  centrifugation  of  the  bacterial  suspen- 
sion in  distilled  water,  obtained  as  in  (i)  with  distilled  water  substituted 
for  the  NaCl  solution. 

(3)  The  NaCl  solution  after   being   applied  to  a  fresh  agar  surface 
and  manipulated  as  though  growth  was  actually  being  removed. 

(4)  The  distilled  water  wash  under  the  same  conditions  as  in  (3). 

is 


(5)  The  supernatant  fluid  from  a  bacterial  suspension  in  a  NaCl  solu- 
tion, the  bacteria  having  been  removed  by  the  spatula  method. 

(6)  The  supernatant  fluid   from  a  bacterial  suspension  in  distilled 
water,  the  growth  having  been  removed  as  in  (5). 

(7)  The  NaCl  solution  used  to  wash  a  glass  slide  manipulated  over  a 
fresh  agar  surface  as  though  growth  was  being  removed. 

(8)  The  distilled  water  wash  obtained  as  in  (7) . 

The  tabulation  below  gives  the  results  of  the  qualitative  tests  for 
phosphate,  chloride,  protein  and  protein  split  products,  and  dextrose. 
The  numbers  in  parentheses  refer  to  the  several  descriptions  listed  above. 


Tests  Performed 

Test  Material 

Protein  or 

Chloride 

Phosphate 

Protein  Split 
Products 

Dextrose 

(i) 

+  +  +  + 

+  +  +  + 

o 

(2) 

+  +  +  + 

-j--J--p-  + 

+  +  +  + 

o 

1 

(5) 

++++ 

± 

0 

o 
o 

0 

(6) 
(7) 

0 

o 

o 
o 

o 
o 

(8) 

o 

o 

0 

o 

Note:  With  medium  containing  I  per  cent,  dextrose  (i)  to  (4)  gave 
for  sugar. 


result 


These  experiments  show  that: 

(1)  Substances  are  transferred  from  the  medium  to  the  wash  solution  . 

(2)  Bacteria  suspended  in  distilled  water  or  NaCl  solution  may  give 
up  some  of  their  substance  to  the  surrounding  liquid. 

(3)  Nothing  is  removed  from  a  medium  by  the  mere  application  of  a 
smooth  spatula  (70). 

This  evidence  points  definitely  to  the  second  method  as  the  superior 
one. 

(d)  Drying  of  growth:  The  technic  was  that  recommended  by  Shackell 
(61)  with  one  exception:  the  bacteria  were  not  frozen  before  drying. 
Layers  I  to  2  mm.  thick  in  shallow  plates  dried  to  a  scaly,  porous  mass 
in  24  to  36  hours.  The  temperature  during  the  process  varied  between  21  ° 
and  22°  C.,  the  pressure  between  o  and  10  mm.  Hg. 

The  material  was  then  pulverized  with  glass  mortar  and  pestle  to  a 
very  fine  powder,  spread  in  thin  layers  on  glazed  paper  plates  and  dried 
to  constant  weight. 

The  dried  substance  was  transferred  to  weighing  bottles  fitted  with 
ground  glass  stoppers  and  stored  in  a  dessiccator  over  concentrated 
H2SO4. 

IV.   ANALYSIS  OF  THE  BACTERIAL  SUBSTANCE 
i.  METHODS: 

(a)  Nitrogen:  Nitrogen  was  determined  by  the  Kjeldahl  process 
(Gunning-Arnold  Dyer  modification)  (62)  .  Digestion  of  the  substance 
in  concentrated  sulphuric  acid  was  completed  with  small  quantities  of 
metallic  mercury.  Before  distillation,  the  mercury  was  precipitated  with 
potassium  sulphide.  The  titrations  were  made  with  0.02  M  NaOH. 

16 


(b)  Total  Sulphur  and  Phosphorus:    Sulphur  was  determined  by  the 
method  adopted  by  the  the  Association  of  Official  Agricultural  Chem- 
ists (63).    The  filtrate  from  the  barium  chloride  precipitation  was  used 
to  determine  phosphorus  (64). 

(c)  Ash:  Inorganic  matter  was  estimated  by  direct  incineration   of 
the  dried  substance  in  a  platinum  crucible  over  a  very  low  flame. 

(d)  Moisture:  The  medium  was  cooled  slowly  to  43°  C.    The  agar  for 
the  entire  series  of  determinations  was  obtained  from   the  same  flask 
and  the  necessary  number  of  petri  dishes  filled  at  the  same  time.    The 
organisms  were  grown  and  collected  as  described  above.    About  o.i  gram 
of  growth  was  rapidly  transferred  to  a  rubber  stoppered  glass  vial  weigh- 
ing about  0.3  gram.    Two  sets  of  determinations  of  water  content  were 
made:  (i)  by  drying  in  vacuo  (o-iomm.  Hgand  22°  C.)  and  (2)  by  drying 
in  an  air  bath  (ioo°-uo°). 

(e)  Lipins  (material  extracted  with  ether) :    The  substance  was  weighed 
in  cone-shaped  filter  paper  thimbles.    The  thimbles  were  suspended  by 
means  of  wires  to  reflux  condensers.    These  were  fitted  with  flasks  con- 
taining ether.    After  24  hours  of  hot  extraction  the  thimbles  containing 
the  extracted  bacteria  were  dried  in  vacuo  to  constant  weight.     The 
difference  in  the  weight  of  the  thimble  before  and  after  extraction  rep- 
presents  the  weight  of  lipins. 

(f)  Blanks:  Blank  determinations  on  the  reagents  used  were  made  in 
all  cases.    In  the  analyses  for  N  the  accuracy  of  the  method  for  the  small 
quantities  obtained  was  checked  by  estimating  the  N  recovered  from 
known  quantities  of  pure  salt  (ammonium  sulphate) . 

2.  RECORDS  OF  ANALYSIS: 

(a)  Organism  No.  688: 

Nitrogen:  0.0199  gram  material  gave  0.0019  gram  N  =  9.55 

per  cent  N;  0.0217  gram  material  gave  0.0022  gram  N  =  10.13 

per  cent  N. 
Sulphur:  0.5336  gram  material  gave  0.0247  gram  BaSO4  =  0.63 

per  cent  S;  0.5769  gram  material  gave  .0259  gram  BaSO4  =  0.61 

per  cent  S. 
Phosphorus:  0.5336  gram  material  gave  0.0393  gram  Mg2P2O7  = 

2.05   per  cent   P;  0.5769   gram   material  gave  0.0432  gram 

Mg2P2O7=  2.08  per  cent  P. 
Ash:  0.5354  gram  material  gave  0.0526  gram  Ash  =  9.82  per 

cent  Ash;  0.4694  gram  material  gave  0.0458  gram  Ash  =  9.75 

per  cent  Ash. 
Moisture:  A.  Determined  by  drying  the  bacterial  material  in 

vacuo:  0.066 1  gram  material  gave  0.0485  gram  H2O  =  73.37 

per  cent;  0.0866  gram  material  gave  0.0636  gram  H2O  =  73.44 

per  cent.     B.  Determined  by  drying  the  bacterial  material 

at  1 00°  C:  0.0899  gram  material  gave  0.0671  gram  H2O  = 

74.63  per  cent;  0.0769  gram  material  gave  0.0573  gram  H2O 

=  74.52  per  cent. 
Lipins:  0.3073  gram  bacterial  material  gave  0.0047  gram  Lipins 

=  1.53  per  cent;  0.3343  gram  bacterial  material  gave  0.0055 

gram  Lipins  =  1.64  per  cent. 

(b)  Organism  No.  720: 

Nitrogen:  0.0257  gram  material  gave  0.0026  gram  N  =  io.ii  per 
cent  N;  0.0243  gram  material  gave  0.0025  gram  N  =  10.28 
per  cent  N. 

17 


Sulphur:  0.5741  gram  material  gave  0.0253  gram  BaSO4  =  0.60 

per  cent  S;  0.5320  gram  material  gave  0.0234  gram  BaSO4  = 

0.60  per  cent  S. 
Phosphorus:  0.5741  gram  material  gave  0.0429  gram  Mg2P2O7  = 

2.08  per  cent  P;  0.5320  gram  material  gave  0.0390  gram 

Mg2P2O7  =  2.O4  per  cent  P. 
Ash:  0.2910  gram  material  gave  0.0301  gram  Ash  =  10.34  Per 

cent  Ash;  0.3011   gram  material  gave  0.0311   gram  Ash  = 

10.33  Per  cent  Ash. 
Moisture:  A.  Determined  by  drying  the  bacterial  material  in 

vacuo:    0.0638  gram  material  gave  0.0452  gram  H2O  =  7O.85 

per  cent;  0.0864  gram  material  gave  0.0611  gram  H2O  =  70.72 

per  cent.    B.  Determined  by  drying  the  substance  at  100°  C: 

0.0781  gram  material  gave  0.0560  gram  H2O  =  7i  .70  per  cent; 

0.0897  gram  material  gave  0.0643  gram  H2O  =  7i.68  per  cent. 
Lipins:    0.3377    gram    bacterial    material   gave  0.0035   gram 

Lipins=i.O3  per  cent;  0.3131  gram  bacterial  material  gave 

0.0033  gram  Lipins  =  1.05  per  cent. 

(c)  Organism  No.  904: 

Nitrogen:  0.0235  gram  material  gave  0.0025  gram  N  =  10.64 

per  cent  N;  0.0213  gram  material  gave  0.0022  gram  N  =  10.33 

per  cent  N. 
Sulphur:  0.6347  gram  material  gave  0.0265  gram  BaSO4  =  o.57 

per  cent  S;  0.5096  gram  material  gave  0.0202  gram  BaSO4  = 

0.54  per  cent  S. 
Phosphorus:  0.6347  gram  material  gave  0.0494  gram  Mg2P2O7 

=  2.17  per  cent  P;  0.5096  gram  material  gave  0.0405  gram 

Mg2P2O7  =  2.2i  per  cent  P. 
Ash:  0.5208  gram  material  gave  0.0538  gram  Ash  =  10.33  Per 

cent  Ash;  0.4933  gram  material  gave  0.0508  gram  Ash  =  10.30 

per  cent  Ash. 
Moisture:  A.  Determined  by  drying  the  substance  in  vacuo: 

0.0475  gram  material  gave  0.0347  gram  H2O  =  73.O5;  0.0620 

gram  material  gave  0.0453  gram  H2O  =  73.O7  per  cent.     B. 

Determined  by  drying  the  bacterial  material  at  100°:  0.0530 

gram  material  gave  0.0401  gram  H2O  =  75-66  per  cent;  0.0668 

gram  material  gave  0.0506  gram  H2O  =  75-75  per  cent. 
Lipins:    0.3390    gram    bacterial    material    gave    0.0122   gram 

Lipins  =  3. 59  per  cent;  0.2981  gram  bacterial  material  gave 

0.0108  gram  Lipins  =  3. 62  per  cent. 

(d)  Organism  No.  1210: 

Nitrogen:  0.0209  gram  material  gave  0.0020  gram  N  =  9-57  per 

cent  N;  0.0238  gram  material  gave  0.0023  gram  N  =  9-66  per 

cent  N. 
Sulphur:  0.5301  gram  material  gave  0.0230  gram  BaSO4  =  o.6o 

per  cent  S;  0.5007  gram  material  gave  0.0217  gram  BaSO4  = 

0.60  per  cent  S. 
Phosphorus:  0.5301  gram  material  gave  0.0410  gram  Mg2P2O7 

=  2.15  per  cent  P;  0.5007  gram  material  gave  0.0399  gram 

Mg2P2O7  =  2.22  per  cent  P. 
Ash:  0.2687  gram  material  gave  0.0266  gram  Ash  =  9. 90  per 

cent  Ash;  0.2087  gram  material  gave  0.0206  gram  Ash  =  9. 87 

per  cent  Ash. 

18 


Moisture:  A.  Determined  by  drying  the  bacterial  material  in 
vacuo:  0.0745  gram  material  gave  0.0544  gram  H2O  =  73.O2 
per  cent;  0.1033  gram  material  gave  0.0753  gram  H2O  =  72.89 
per  cent.  B.  Determined  by  drying  the  bacterial  material  at 
100°:  0.0466  gram  material  gave  0.0349  gram  H2O  =  74-9O 
per  cent;  0.0739  gram  material  gave  0.0554  gram  H2O  =  74.97 
per  cent. 

Lipins:  0.2051  gram  bacterial  material  gave  0.0083  gram  Lipins 
=  4.04  per  cent;  0.2251  gram  bacterial  material  gave  0.0086 
gram  Lipins  =  3. 82  per  cent. 

(e)  Organism  No.  863: 

Nitrogen:  0.0265   gram   material   gave  0.0026   gram   N  =  9.8i 

per  cent  N;  0.0224  gram  material  gave  0.0023  gram  N  =  10.27 

per  cent  N. 
Sulphur:  0.6206  gram  material  gave  0.0254  gram  BaSO4  =  o.56 

per  cent  S;  0.7039  gram  material  gave  0.0283  gram  BaSO4  = 

0.55  per  cent  S. 

Phosphorus:  0.6206  gram  material  gave  0.0444  gram  Mg2P2O7  = 

1.99  per  cent  P;  0.7039  gram  material  gave  0.0498  gram 

Mg2P2O7=i.97  per  cent  P. 
Ash:  0.4887  gram  material  gave  0.0457  gram  Ash  =  9.35  per  cent 

Ash;  0.5881  gram  material  gave  0.0552  gram  Ash  =  9. 38  per 

cent  Ash. 

Moisture:  A.  Determined  by  drying  the  bacterial  material  in 
vacuo:  0.0665  gram  material  gave  0.0465  gram  ^0  =  69.93 
per  cent;  o.  0743  gram  material  gave  0.0521  gram  H2O  =  7O.I2 
per  cent.  B.  Determined  by  drying  the  substance  at  100°: 
°-°577  gram  material  gave  0.0432  gram  H2O  =  74.87  per  cent. 

Lipins:  0.4098  gram  bacterial  material  gave  0.0226  gram  Lipins 
=  5.51  per  cent;  0.3933  gram  material  gave  0.0208  gram  Lipins 
=  5.29  per  cent. 

(f)  Organism  No.  1074: 

Nitrogen:  0.0209  gram  material  gave  0.0021   gram  N=  10.05 

per  cent  N;  0.0198  gram  material  gave  0.0019  gram  N  =  9-59 

per  cent  N. 
Sulphur:  0.5037  gram  material  gave  0.0221  gram  BaSO4  =  o.6o 

per  cent  S;  0.4791  gram  material  gave  0.0199  gram  BaSC>4  = 

0.57  per  cent  S. 

Phosphorus:  0.5037  gram  material  gave  0.0358  gram  Mg2P2O7 
=  1.98  per  cent  P;  0.4791  gram  material  gave  0.0338  gram 
Mg2P2O7=  1.96  per  cent  P. 

Moisture:  A.  Determined  by  drying  the  bacterial  material  in 
vacuo:  0.1070  gram  material  gave  0.0765  gram  H2O  =  7i-5O 
per  cent;  0.0896  gram  material  gave  0.0642  gram  H2O  =  7i.65 
per  cent.  B.  Determined  by  drying  the  substance  at  100°: 
0.0668  gram  material  gave  0.0506  gram  H2O  =  75.60  per  cent; 
0.0552  gram  material  gave  0.0416  gram  H2O  =  75-36  per  cent. 

Lipins:  0.3080  gram  bacterial  material  gave  0.0166  gram  Lipins 
=  5-39  Per  cent;  0.2995  gram  bacterial  material  gave  0.0162 
gram  Lipins  =  5. 40  per  cent. 

Ash:  0.3024  gram  material  gave  0.0301  gram  Ash  =  9. 95  per 
cent  Ash;  0.4024  gram  material  gave  0.0398  gram  Ash  =  9. 89 
per  cent  Ash. 

19 


3.  SUMMARY  OF  THE  DATA  FOR  THE  AVERAGE  PERCENTAGE  COMPOSI- 
TION OF  THE  STREPTOCOCCI. 


Description  of  Organism 

Composition 

Moisture 

Nu  mber 

Type 

N 

S 

P 

Ash 

Lipins 

Vacuo 

IOO°-1  10° 

688 

Indifferent 

9.8 

0.62 

2.06 

9.78 

1.58 

7340 

74-57 

720 

Indifferent 

IO.I 

0.60 

2.06 

10.33 

1.04 

70.78 

71.69 

904 

Hemolytic 

10.4 

0-55 

2.19 

10.31 

3.60 

73.06 

75-70 

I2IO 

Hemolytic 

9.6 

0.60 

2.18 

9.88 

3-93 

72.95 

74-93 

863 

Green  producing 

IQ.O 

0-55 

1.98 

9-36 

5-40 

70.02 

74-87 

1074 

Green  producing 

9.8 

0.58 

1.97 

9.92 

5-39 

71-57 

7548 

4.  CONCLUSIONS  FROM  THE  ABOVE  ANALYTICAL  DATA:  The  above 
summary  indicates  the  average  results  of  the  various  analyses.  It  also 
brings  into  comparison  the  figures  for  the  composition  of  the  different 
streptococci  and  points  out  these  facts: 

(a)  There  are  no  marked  graded  differences  in  either  the  nitrogen  or 
the  sulphur  content.     It  becomes  apparent,  therefore,  that  the  total 
amounts  of  protein  material  in  the  different  organisms  varies  but  slightly, 
if  at  all. 

(b)  The  phosphorus  content  is  practically  constant.    This  indicates  no 
striking  variations  in  the  quantity  of  nucleoprotein  (the  phosphorus  due 
to  phosphatide  being  very  small  in  amount) . 

(c)  Regarding  the  results  for  the  moisture  determinations  only  slight 
deviations  are  noticed.     The  percentages  obtained  by  drying  the  sub- 
stance at  ioo°C.  are  higher  than  those  obtained  by  drying  the  material 
in  vacuo.     Such  increases  in  the  values  were  expected  because  of  the 
greater  disintegration  at  the  higher  temperature. 

(d)  Most  interesting,  however,  are  the  figures  obtained  for  the  amounts 
of  Lipin  material.     These  vary  greatly  for  the  different  types  of  strep- 
tococci.    The  percentage  of  lipins  is  lowest  in  the  indifferent  type  and 
highest  in  the  green  producing  type,  the  hemolytic  type  taking  a  posi- 
tion between  these  two.     The  characteristic  relations   described  above 
may  be  expressed  by  the  ratio:     10     :     29     :     41     ;     or     briefly     : 


Indifferent: 


Hemolytic:  Green-producing 
3:4 


These  results  separate  the  streptococci  into  three  groups  corre- 
sponding to  those  obtained  when  the  reaction  on  blood  media  is  used  as 
the  criterion. 

Because  of  the  apparent  significance  of  this  relationship,  we  repeated 
this  part  of  the  investigation.  Fresh  cultures  were  amassed  and  dried 
as  described  above.  A  series  of  nine  organisms  were  used,  three  from 
each  representative  group,  the  strains  688,  720,  904,  1210,  863  and  1074 
being  included  .  The  three  new  strains  were  chosen  at  random  from  our 
stock  cultures  of  indifferent,  green-producing  and  hemolytic  streptococci. 

These  results  were  obtained: 


20 


Designation  of  Organism 

Number  Type  Average  Percentage  Lipins 

688  Indifferent  1.46 

720  Indifferent  1 .54 

725  Indifferent  1.16 

904  Hemolytic  3.16 

1210  Hemolytic  3.73 

1012  Hemolytic  3-79 

863  Green-producing  5.31 

1074  Green-producing  5.34 

953  Green-producing  5.68 

A  mere  glance  at  the  table  brings  to  light  the  possible  ratio: 

1:3:4 
This  similates  the  data  gathered  from  our  first  group  of  determinations. 

V.   ANALYSIS  OF  THE  LIPIN  MIXTURE 

1.  OUTLINE   FOR   THE   WORK:    The  very  striking  regularity   in   the 
amounts  of  Lipin  material  and  the  fact  that  the  groups  of  streptococci 
parallel  those  obtained  by  the  blood  media  reaction  suggested  the  possible 
relation  between  the  nature  of  the  Lipin  mixture  and  the  hemolyzing 
capacity  of  the  organisms.    In  an  investigation  of  this  character  the  chief 
factors  to  be  considered  are  (i)  the  phosphatide  content,  (2)  the  choles- 
terol content  and  (3)  the  ratio  between  the  phosphatide  and  cholesterol. 

A  preliminary  qualitative  examination  of  the  material  showed  that  all 
the  extracts  contained  both  cholesterol  and  phosphatide.  These  sub- 
stances were  then  determined  quantitatively  by  nephelometric  methods 
(65,66,67,68). 

2.  RECORDS  OF  ANALYSIS  OF  THE  LIPIN  MIXTURE. 

(a)  Organism    No.    688: 

Phosphatide:  2.3560  gram  bacterial  material  gave  0.0400  gram 
Lipins  which  yielded  0.0000125  gram  H3PO4=  0.25  per  cent 
Phosphatide;  2.4960  gram  bacterial  material  gave  0.0380  gram 
Lipins  which  yielded  0.0000125  gram  H3PO4=  0.25  per  cent 
Phosphatide. 

Cholesterol:  2.5360  gram  bacterial  material  gave  0.0400  gram 
Lipins  which  yielded  0.00064  gram  Cholesterol  =1.6  percent 
Cholesterol;  2. 4960 gram  bacterial  material  gave  0.0380  gram 
Lipins  which  yielded  0.00064  gram  cholesterol  =  1.7  per  cent 
cholesterol. 

(b)  Organism  No.  720: 

Phosphatide:  2.4411  gram  bacterial  material  gave  0.0290  gram 
Lipins  which  yielded  0.0000087  gram  H3PO4  =  0.24  per  cent 
Phosphatide;  2.4301  gram  bacterial  material  gave  0.0280 
gram  Lipins  which  yielded  0.0000087  gram  H3PO4=  0.24 
per  cent  Phosphatide. 

Cholesterol:  2.4411  gram  bacterial  material  gave  0.0290  gram 
Lipins  which  yielded  0.00046  gram  Cholesterol  =  1.5  per  cent 
2.4302  gram  bacterial  material  gave  0.0280  gram  Lipins  which 
yielded  0.00046  gram  Cholesterol  =1.5  per  cent. 

(c)  Organism  No.  904: 

Phosphatide:  1.7090  gram  bacterial  material  gave  0.0710  gram 
Lipins  which  yielded  0.000006  gram  H3PO4  =  0.064  per  cent 
Phosphatide;  i  .6980  gram  bacterial  material  gave  0.0640  gram 


Lipins    which  yielded  0.000006  gram  H3PO4=  0.072  per  cent 
Phosphatide. 

Cholesterol:  1.7090  gram  bacterial  material  gave  0.0710  gram 
Lipins  which  yielded  0.00078  gram  Cholesterol  =  1.09  per 
cent;  1.6980  gram  bacterial  material  gave  0.0640  gram  Lipins 
which  yielded  0.0007  gram  Cholesterol  =  1.08  per  cent. 

(d)  Organism  No.  1210: 

Phosphatide:  1.7091  gram  bacterial  material  gave  0.0700  gram 
Lipins  which  yielded  0.000006  gram  HsPO4  =  0.064  per  cent 
Phosphatide;  1.7100  gram  bacterial  material  gave  0.0710 
gram  Lipins  which  yielded  0.000006  gram  H3PO4  =  0.064 
per  cent  Phosphatide. 

Cholesterol:  1.7091  gram  bacterial  material  gave  0.0700  gram 
Lipins  which  yielded  0.0007  gram  Cholesterol  =  i.o  per  cent 
Cholesterol;  1.7100  gram  bacterial  ma'terial  gave  0.0710  gram 
Lipins  which  yielded  0.00074  gram  Cholesterol  =  1.02  per 
cent. 

(e)  Organism  No.  86  j: 

Phosphatide:  2.5725  gram  bacterial  material  gave  0.1366  gram 

Lipins  which  yielded  0.000006  gram  H3PO4=  0.035  Per  cent 

Phosphatide;  2.5728  gram  bacterial  material  gave  0.1362  gram 

Lipins  which  yielded  0.000006  gram  H3PO4  =  0.035  per  cent 

"  Phosphatide. 

Cholesterol:  2.5725  gram  bacterial  material  gave  0.1366  gram 
Lipins  which  yielded  o.ooio  gram  Cholesterol  =  0.73  per  cent; 
2.5728  gram  bacterial  material  gave  0.1362  gram  Lipins 
which  yielded  o.ooio  gram  Cholesterol  =  0.73  per  cent. 

(f)  Organism  No.  1074: 

Phosphatide:  2.5361  gram  bacterial  material  gave  0.1321  gram 
Lipins  which  yielded  0.000006  gram  H3PO4  =  0.036  per  cent 
Phosphatide;  2.5360  gram  bacterial  material  gave  0.1325  gram 
Lipins  which  yielded  0.000006  gram  H3PO4  =  0.036  per  cent 
Phosphatide. 

Cholesterol:  2.5361  gram  bacterial  material  — *~  ~  """ 

Lipins  which  yielded  o.ooio  gram  Cholester 


3.  SUMMARY  OF  THE  DATA  FOR  THE  AVERAGE  PERCENTAGES  OF  PHOS- 
PHATIDE   AND    CHOLESTEROL. 


Organism  No. 

688 

720 

904 

1210 

863 

1074 

Phosphatide 

0.25 

0.24 

0.068 

0.064 

0.035 

0.036 

Cholesterol 

l.65 

1.50 

1.  08 

I.OI 

0.73 

0.75 

Ratio  C:P 

6:1 

6:1 

16:1 

16:1 

21:1 

21:1 

4.  CONCLUSIONS  FROM  THE  DATA  FOR  THE  CHOLESTEROL  AND  PHOS- 
PHATIDE CONTENT. 

(a)  The  content  of  phosphatide  decreases  in  the  different  types  in  the 
order  named:  indifferent,  hemolytic,  green-producing. 

(b)  The  same  relation  is  noticed  in  the  amounts  of  cholesterol  contained 
in  the  different  organisms. 

(c)  The  ratio  C:P  increases  when  the  types  are  listed  in  the  same  order. 


VI.    EFFECT  OF  THE  LIPIN  MATERIAL  ON  HEMOLYSIS 

i.  RECORD  OP  EXPERIMENTS:  Since  the  ratios  of  cholesterol  to  phos- 
phatide  are  different  for  the  different  types  of  streptococci  it  was  interest- 
ing to  investigate  the  effects  which  the  lipin  mixtures  would  have  on  the 
hemolytic  action  of  saponin.*  A  10  per  cent  washed  rabbit  cell  suspen- 
sion was  used  in  the  experiments.  The  method  used  was  essentially  that  of 
Ranson  (69).  Preliminary  titrations  were  made  to  obtain  the  dose  of 
saponin  that  will  hemolyze  I  .o  c.c.  of  cell  suspension  in  the  presence  and 
absence  of  cholesterol  (the  amount  of  cholesterol  used  approximated  that 
in  the  lipin  mixture) .  That  amount  of  saponin  which  produced  hemolysis 
in  the  absence  of  cholesterol  but  which  failed  to  do  so  in  those  tubes  which 
contained  cholesterol  was  taken  as  the  test  dose.  This  dose  was  then 
added  to  graded  amounts  of  lipin  material  dissolved  in  ether.  The  pro- 
tocols below  show  in  detail  the  experiments  performed  and  the  results 
obtained. 

Experiment  (a):  To  determine  the  largest  amount  of  saponin  that 
produces  hemolysis  in  the  presence  of  cholesterol. 


Tube  no. 

i 

2 

3 

4 

5 

6 

7 

8 

9 

Saponin  in  c.c. 
0.2%  in  0.9% 
NaCl 

I.O 

0.5 

0.2$ 

O.IS 

O.IO 

O.08 

0.06 

0.04 

o.o* 

Cholesterol  in  c.c. 
0.01%  in  Ether 

O.  I 

0.  I 

O.  I 

O.  I 

O.  I 

O.  I 

O.I 

O.  I 

0.  I 

0.9%  NaCl  inc.  c. 

1.90 

2.40 

2.63 

2.7S 

2.  SO 

2.82 

2.84 

a.  86 

s.88 

Incubation  at  25°  C.  for  15  Minutes 


Cell  suspension  in 
c.c. 

I.O 

I.O 

I.O 

I.O 

I.O 

I.O 

I.O 

I.O 

I.O 

Readings: 

8  minutes 

+  +  +  + 

+  +  +  + 

++++ 

+++ 

+++ 

o 

o 

0 

0 

15  minutes 

+  +  +  + 

+  +  +  + 

++++ 

+++ 

+++ 

+ 

+ 

o 

o 

60  minutes 

+  +  +  + 

+  +  +  + 

+++  + 

+++  + 

++++ 

+  +  + 

+  + 

o 

o 

12  hours 

+  +  +  + 

+  +  +  + 

++++ 

+++  + 

+++  + 

+  +  +  + 

+  +  +  + 

++++ 

o 

This  experiment  shows  that  in  the  presence  of  cholesterol  0.08  cc.  of 
a  0.2%  saponin  solution  is  the  largest  amount  that  does  not  hemolyze 
i.o  c.c.  cell  suspension  in  8  minutes. 

Experiment  (b):  To  determine  the  smallest  amount  of  saponin  that 
produces  hemolysis  in  the  absence  of  cholesterol . 


Tube  no. 

i 

2 

3 

4 

5 

6 

7 

8 

0 

Saponin 

I.O 

0.5 

0.25 

0.15 

0.  I 

0.08 

0.06 

0.04 

o.  02 

Ether 

O.  I 

O.  I 

O.  I 

O.  I 

O.  I 

O.  I 

O.  I 

0.  I 

0.  I 

Salt  Solution 

1.90 

2.40 

2.65 

2.75 

2.80 

2.82 

2.  84 

2.8. 

2.8* 

Incubation  at  25°  C.  for  15  Minutes 

Cell  Suspension          i.o  i.o  i.o  i.o  i.o  i.o  i.o  i.o          i.o 

Readings: 
8  minutes 

15  minutes 

60  minutes 

12  hours 

*The  saponin  was  obtained  from  Eimer  and  Amend,  New  York  City. 

23 


The  results  of  this  experiment  indicate  that  in  the  absence  of  choles- 
terol 0.08  c.c.  of  a  0.2%  saponin  solution  is  the  smallest  amount  that 
produces  hemolysis  with  i.o  c.c.  cell  suspension  in  8  minutes.  This  is, 
therefore,  the  test  dose  and  was  used  in  determining  the  effect  of  the 
ether  extract  on  the  action  of  saponin. 

The  lipins  extracted  from  the  bacteria  were  dissolved  in  definite  volumes 
of  ether:  three-fifths  of  this  solution  was  evaporated  to  a  volume  of  3cc. 
and  then  emulsified  with  27  cc.  of  0.9%  NaCl  solution.  Graded  amounts 
of  this  emulsion  were  added  to  the  various  tubes. 

Experiment  (c):  To  determine  the  effect  of  the  extract  from  organism 
no.  688  on  the  action  of  saponin. 


Tube  No. 

I 

2 

3 

4 

Extract  in  c.c. 

I.O 

i-5 

2.0 

2-5 

Saponin  in  c.c. 

0.08 

0.08 

0.08 

0.08 

Salt  solution  in  c.c. 

1.92 

1.42 

O.92 

0.42 

Incubation  at  25°  for  15  Minutes 


Cell  suspension  in  c.c. 

I.O 

I.O 

I.O 

I.O 

Readings: 
10  minutes 

0 

o 

0 

o 

20  minutes 

o 

o 

o 

o 

I  hour 

-j_ 

-j_ 

-)_ 

.f. 

12  hours 

+ 

+ 

+ 

4- 

Experiments  (d),  (e),  (f),  (g),  (h)  were  performed  respectively  with 
extracts  from  organisms  720,  904,  1210,  863,  1074.  The  results  parallelled 
exactly  those  of  experiment  (c). 

As  checks  on  the  validity  of  the  above  experiments  controls  were  run 
on  the  various  ingredients: 


No. 

i 

2 

3 
4 
5 
6 

7 
8 

9 
10 


Combination 

Salt  solution  and  cell  suspension 
Lecithin  and  cell  suspension 
Cholesterol  and  cell  suspension 
Saponin  and  cell  suspension 
Extract  688  and  cell  suspension 
Extract  720  and  cell  suspension 
Extract  904  and  cell  suspension 
Extract  1210  and  cell  suspension 
Extract  863  and  cell  suspension 
Extract  1074  and  cell  suspension 


Hemolysis 
o 
o 
o 

4-4-4-  + 
o 
o 
o 
o 
o 
o 


2.  CONCLUSIONS  FROM  DATA  ON  HEMOLYSIS. 


(a)  The  lipin  mixtures  from  the  various  organisms  show  the  same 
effect  on  the  action  of  saponin — they  inhibit  hemolysis. 

(b)  The  differences,  mentioned  above,  in  the  ratios  of  cholesterol  to 
phosphatide  cannot  be  correlated  with  differences  in  the  action  of  the 
ether  extracts  on  the  hemolytic  power  of  saponin. 


VII.   SUMMARY  OF  CONCLUSIONS. 

1 .  There  are  no  marked  graded  differences  in  either  the  N  or  S  content. 
It  becomes  apparent,  therefore,  that  the  total  amounts  of  protein  material 
in  the  different  organism  varies  but  slightly. 

2.  The  P  content  is  practically  constant.     This  indicates  no  striking 
variations  in  the  quantity  of  nucleoprotein  (the  P  from  the  phosphatide 
being  very  small  in  amount). 

3.  Regarding  the  results  for  the  moisture  determinations  only  slight 
deviations  are  noticed.    The  percentages  obtained  by  drying  the  material 
at  100°  C.  are  higher  than  those  obtained  by  drying  the  substances  in 
vacuo.    Such  increases  in  the  values  were  expected  because  of  the  greater 
disintegration  at  the  higher  temperature. 

4.  Most  interesting  are  the  figures  obtained  for  the  amount  of  lipin 
material.     These  vary  greatly  for  the  different  types  of  streptococci. 
The  percentage  of  lipins  is  lowest  in  the  indifferent  type  and  highest  in 
the  green-producing  type,  the  hemolytic  type  taking  a  position  between 
these  two.    The  characteristic  relations  may  be  expressed  by  the  ratio: 
Indifferent:  Hemolytic:  Green  Producing::   1:3:4.    These  results  seem 
to  seperate  the  streptococci  into  three  groups  corresponding  to  those 
obtained  when  the  reaction  on  blood  is  used  as  the  criterion. 

5.  The  content  of  phosphatide  in  the  lipin  mixture  decreases  for  the 
different  types  in  the  order  named:  indifferent,  hemolytic,  green-pro- 
ducing. 

6.  The  same  relation  is  given  by  the  amounts  of  cholesterol  contained 
in  the  different  organisms. 

7.  The  ratio  C:P  increases  when  the  types  are  listed  as  above. 

8 .  The  lipin  mixtures  from  the  various  organisms  show  the  same  effect 
on  the  action  of  saponin:  they  inhibit  hemolysis. 

9.  The  above  mentioned  differences  in  the  ratios  of  cholesterol  to 
phosphatide  cannot  be  correlated  with  differences  in  the  action  of  the 
ether  extracts  on  the  hemolytic  power  of  saponin. 


25 


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27 


ACKNOWLEDGMENT 

Throughout  the  course  of  these  researches 
I  have  received  many  important  suggestions 
from  Professor  W.  J.  Gies,  Dr.  E.  G.  Miller, 
Jr.,  and  the  several  members  of  the  Depart- 
ment of  Biological  Chemistry. 

It  is,  therefore,  a  pleasure  to  express  my 
extreme  indebtedness  for  the  valuable  criticism 
and  helpful  guidance  which  were  so  willingly 
given  me. 


PUBLICATION 

Is  there  a  Parallelism  Between  Normal  Agglutinins  and  Hemolysins  of 

Human  Blood 

(with  Reuben  Ottenberg) 

Proceedings  of  the  New  York  Pathological  Society, 
1919,  xix,  83 


VITA 

Frances  Krasnow  was  born  in  New  York,  October  16,  1894.  She 
received  her  preliminary  and  secondary  education  in  the  Public  Schools 
of  New  York  City.  In  December  1912  she  was  elected  to  free  member- 
ship in  the  Brooklyn  Institute  of  Arts  and  Sciences.  From  February  to 
June  1913  she  pursued  studies  at  this  Institute  and  at  Hunter  College. 
Receiving  a  University  Scholarship  in  August  1913  she  entered  Barnard 
College,  Columbia  University,  and  was  graduated  with  the  degree  Bache- 
lor of  Science,  magna  cum  laude,  in  February  1917.  She  continued  her 
work  in  the  Graduate  Schools  and  received  the  degree  of  Master  of  Arts 
in  June  1917.  At  the  same  time  she  was  awarded  the  Kohn  prize  for 
Mathematics,  given  honors  for  Zoology  and  Physics  and  elected  member 
of  the  Phi  Beta  Kappa  Society. 

In  July  1918  she  was  appointed  assistant  in  Bacteriology  at  Columbia 
University,  and  in  the  Fall  of  1919  received  a  similar  appointment  in  the 
Department  of  Biological  Chemistry,  which  position  she  now  holds. 


Gaylord  Bros. 

Makers 

Syracuse,  N.  Y. 
PAT.  JAN.  21, 1908 


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